2-Aryl-4-Quinazolinones And Their Pharmaceutical Compositions

ABSTRACT

Provided is a compound of the formula I or a pharmaceutically acceptable salt, solvate or stereoisomer thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein Ar represents 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             R 5 , R 6 , R 7 , R 8,  R 1 ′, R 2 ′, R 3 ′, R 4 ′, R 5 ′, R 6 ′, R 7 ′, R 8 ′ independently represent H, OH, F, Cl, Br, C 1  to C 6  alkyl group, C 1  to C 6  alkoxy group, C 2  to C 6  alkenyl group, C 2  to C 6  alkenoxy group, C 2  to C 6  alkynyl group, C 2  to C 6  alkynoxy group, amine group, mono- or di-substituted amino group, cyclic C 1  to C 5  alkylamino group, imidazolyl group, morpholino group, piperazinyl group, optionally substituted with one or more hydroxy or halo; and X represents NH, O or S. The present invention also provides a composition comprising the compound of formula I. The compound and the composition in accordance with the present invention are effective on treating or alleviating a disease or disorder, such as malignant glioma.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to 2-aryl-4-quinazolinones andpharmaceutical compositions comprising the same for treating cancer,particularly for malignant gliomas.

2. Description of the Prior Arts

Primary brain and spinal cord tumors are among the top 10 causes ofdeaths due to cancer with over 16,000 people dying from them each yearin the United States. Malignant gliomas account for approximately 70% ofthe 22,500 new cases of malignant primary brain tumor that are diagnosedin adults in the United States each year. Although the prevalence ofmalignant glioma is relative lower than that of the other cancers suchas lung, breast, prostate, colorectal, and liver cancers, malignantglioma has high mortality. In Taiwan, the frequencies of death ofmalignant brain tumor hit 1.06% in men and 1.45% in women, ranked the15^(th) and 13^(th) respectively. Glioblastoma multiforme (GBM) definedby WHO as grade IV is the most common and most aggressive type of gliomain humans. Despite optimal treatment, the median survival is only 12 to15 months for patients with glioblastomas. Despite recent advances insurgery, radiotherapy, and chemotherapy, the prognosis of patientsdiagnosed with malignant gliomas remains very poor. The currenttechnique lacks a compound with profound efficacy on gliomas. Therefore,the development of the new anti-malignant glioma drugs is urgentlyneeded.

SUMMARY OF THE INVENTION

Given the need of solution for gliomas, the objective of the presentinvention is to provide a novel compound and composition that haveprofound efficacy on gliomas.

Accordingly, the present invention provides a compound of the formula

wherein R₅, R₆, R₇ and R₈ independently represent H, OH, F, Cl, Br, C₁to C₆ alkyl group, C₁ to C₆ alkoxy group, C₂ to C₆ alkenyl group, C₂ toC₆ alkenoxy group, C₂ to C₆ alkynyl group, C₂ to C₆ alkynoxy group,amine group, mono- or di-substituted amino group, cyclic C₁ to C₅alkylamino group, imidazolyl group, morpholino group, piperazinyl group,optionally substituted with one or more hydroxy or halo; Ar represents

R₁′, R₂′, R₃′, R₄′, R₅′, R₆′, R₇′, R₈′ independently represent H, OH, F,Cl, Br, C₁ to C₆ alkyl group, C₁ to C₆ alkoxy group, C₂ to C₆ alkenylgroup, C₂ to C₆ alkenoxy group, C₂ to C₆ alkynyl group, C₂ to C₆alkynoxy group, amine group, mono- or di-substituted amino group, cyclicC₁ to C₅ alkylamino group, imidazolyl group, morpholino group,piperazinyl group, optionally substituted with one or more hydroxy orhalo; X represents NH, O or S; or a pharmaceutically acceptable salt,solvate or stereoisomer thereof.

In another aspect, the present invention provides a compositioncomprising a compound provided herein and a carrier.

In another aspect, the present invention provides a method for treatingdiseases or disorders comprising: administering the compound of theformula I or composition comprising the same to a subject in needthereof.

Other objectives, advantages and novel features of the invention willbecome more apparent from the following detailed description when takenin conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of MJ-66 on cell viability in HT29-5FURcells;

FIG. 2 illustrates concentration-dependent effects of MJ-66, MJ-68 andMJ-78 on glioma cell lines including C6 and U87 for 48 hr (mean±SEM,n=3), wherein C6 represents rat glioma cells and U87 represents humanglioma cells;

FIG. 3A illustrates inhibition of glioma cell proliferation by MJ-66 inconcentration- and time-dependent way, wherein the used target cellswere glioma cell lines C6, RT2, U87, U373, U251 and T98G as indicated;

FIG. 3B illustrates that glioma cell survival was reduced aftertreatment with 60 nM MJ-66 time-dependently by MTS assay (mean±SEM,n=3), wherein rat glioma cell lines C6 and RT2 and human glioma celllines U87, U373, U251 and T98G were respectively used as target cells asidentified; and

FIGS. 4A and 4B illustrate that MJ-66 induced glioma cell death; FIG. 4Ademonstrates the morphology of C6 and U87 glioma cell lines after beingtreated with 30, 60, 90 nM MJ-66 or 0.01% DMSO for indicated time under200× bright field microscope; FIG. 4B demonstrates the responses of C6and U87 glioma cells after being treated with 60 nM MJ-66 or vehicle(medium or DMSO) for different time durations (mean±SEM, n=3) (*p<0.05versus control);

FIGS. 5A and 5B illustrate that MJ-66 induced glioma G2/M arrest andcell death; FIG. 5A demonstrates that cell cycle distributions of C6glioma cells being treated with 60 nM MJ-66 for indicated time; FIG. 5Bis the graph displaying the percentages of subG1, G1, S, G2/M and >4NDNA content analyzed from FIG. 5A;

FIGS. 6A, 6B and 6C demonstrate that MJ-66 did not causenon-proliferating glioma cell to undergo cell death;

FIG. 6A illustrates the effects of serial concentrations of MJ-66 onhuman normal glia cell line (SVGP12) and rat primary glia cells (Glia)for different time durations, which are determined by MTS assay;

FIG. 6B is the result from flow cytometric analysis of C6 glioma cellstreated with 41.6 nM Ara-C for 12 hr;

FIG. 6C demonstrates, in the upper panel, the results of C6 glioma cellstreated with 60 nM MJ-66 for indicated time; and, in the middle andlower panels, the results of Ara-C-cotreated C6 cells being treated withsolvent (DMSO) or MJ-66 for indicated time;

FIGS. 7A, 7B and 7C demonstrate that MJ-66-induced multinucleatedphenotype and multipolar spindles in glioma cells;

FIG. 7A provides representative images showing abnormal mitoticU87glioma cells after treated with 60 nM MJ-66 for 24 hr stained withHoechst 33342 and antibody for alpha-tubulin (green) and U87 gliomacells treated with 0.006% DMSO in medium as control;

FIG. 7B provides the confocal laser scanning microscopy of the cellstreated with 0.006% DMSO in medium or 60 nM MJ-66 for 12 hr stained withalpha-tubulin (red) and gamma-tubulin (green), wherein arrows aredirected to multinucleated cells; arrow heads are directed to multipolarspindle; and white scale bar is equivalent to 10 μm;

FIG. 7C is the result of quantitative analysis of multinucleated cellsafter treated with 60 nM MJ-66 for various time durations;

FIG. 8 demonstrates MJ-66 inhibited tumor growth in xenograft animalmodel (**p<0.01), versus control and DMSO.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment, provided is 2-aryl-4-quinazolinone or apharmaceutically acceptable salt, solvate or stereoisomer thereof.

In another embodiment, provided is a composition, which comprises the2-aryl-4-quinazolinone or a pharmaceutically acceptable salt, solvate orstereoisomer thereof; and a carrier.

In another embodiment, provided is a method for treating or alleviatingdiseases or disorders, comprising administering the2-aryl-4-quinazolinone or its pharmaceutically acceptable salt, solvateor stereoisomer to a subject in need thereof.

In a preferred embodiment, the 2-aryl-4-quinazolinone is the compound ofthe formula I:

wherein R₅, R₆, R₇ and R₈ independently represent H, OH, F, Cl, Br, C₁to C₆ alkyl group, C₁ to C₆ alkoxy group, C₂ to C₆ alkenyl group, C₂ toC₆ alkenoxy group, C₂ to C₆ alkynyl group, C₂ to C₆ alkynoxy group,amine group, mono- or di-substituted amino group, cyclic C₁ to C₅alkylamino group, imidazolyl group, morpholino group, piperazinyl group,optionally substituted with one or more hydroxy or halo; Ar represents

R₁′, R₂′, R₃′, R₄′, R₅′, R₆′, R₇′, R₈′ independently represent H, OH, F,Cl, Br, C₁ to C₆ alkyl group, C₁ to C₆ alkoxy group, C₂ to C₆ alkenylgroup, C₂ to C₆ alkenoxy group, C₂ to C₆ alkynyl group, C₂ to C₆alkynoxy group, amine group, mono- or di-substituted amino group, cyclicC₁ to C₅ alkylamino group, imidazolyl group, morpholino group,piperazinyl group, optionally substituted with one or more hydroxy orhalo; and X represents NH, O or S.

The term “C₁ to C₆ alkyl” as used herein refers to a branched orunbranched alkyl group having 1 to 6 carbon atoms. For example, suchgroup is methyl, ethyl, isopropyl, tertiary-butyl, pentyl or hexylgroup. Similarly the term “C₁ to C₂ alkyl” as used herein refers to analkyl group having 1 to 2 carbon atoms.

The term “C₂ to C₆ alkenyl” as used herein refers to a branched orunbranched alkenyl group having 2 to 6 carbon atoms and at least onedouble bond. For example, such group is ethenyl or isopropenyl.

The term “C₂ to C₆ alkynyl” as used herein refers to a branched orunbranched alkynyl group having 2-6 carbon atoms and at least one triplebond. For example, such group is ethynyl or 3-methyl-1-butylyl.

The term “mono-substituted amine group” as used herein refers to primaryamine group with a branched or unbranched alkyl substitute. Likewise,the term “di-substituted amine group” as used herein refers to secondaryamine group with a branched or unbranched alkyl substitute. In apreferred embodiment, di-substituted amino group is dimethylamine group.

Preferably, said cyclic C₁ to C₅ alkylamino group includes, but notlimited to, pyridinyl group, pyrrolidinyl group, piperidinyl group andmorpholino group.

In one embodiment, Ar is naphthalenyl group. In another embodiment, Aris indolyl group. In still another embodiment, Ar is benzothiophenylgroup.

In one embodiment, R₆ is hydrogen group. In another embodiment, R₆ ismethoxyl group. In another embodiment, R₆ is dimethylamino group. Inanother embodiment, R₆ is pyrrolidinyl group. In still anotherembodiment, R₆ is piperidinyl group. In yet another embodiment, R₆ ismorpholino group.

Specific examples of the compound in accordance with the presentinvention are, but not limited to:

The term “pharmaceutically acceptable salt” as used herein refers toacid addition salts prepared from non-toxic acid and base salts preparedfrom non-toxic base. Suitable acids include, but not limited to, acetic,alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethenesulfonic, formic, fumaric, furoic, gluconic, glutamic, glucorenic,galacturonic, glycidic, hydrobromic, hydrochloric, isethionic, lactic,maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phenylacetic, propionic, phosphoric, salicylic, stearic,succinic, sulfanilic, sulfuric, tartaric acid, p-toluenesulfonic and thelike. In one embodiment, suitable acids are hydrochloric, hydrobromic,phosphoric, and sulfuric acids.

Suitable bases include, but not limited to, alkali metal cations such aspotassium and sodium hydroxide; alkaline earth metal cations such ascalcium and magnesium; ammonium or water-soluble amine and the loweralkanolammonium. Particularly, suitable bases include aluminium,arginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, tromethamine and hydroxide of magnesium, meglumine,olamine, potassium, sodium or zinc.

The term “solvate” as used herein refers to a complex of variablestoichiometry formed by a solvent and a solute, which is a compound offormula I in the present invention. Such solvent has little interferencewith the biological activity of the solute. For example, suitablesolvents include water, methanol, ethanol and acetic acid. Preferably,the solvent is a pharmaceutically acceptable solvent, such as water,ethanol and acetic acid.

The compound of the formula I in accordance with the present inventioncan be in all stereoisomeric forms. Said stereoisomeric forms are suchas enantiomers, diastereoisomers, and cis- and trans-isomers. Forexample, in the case where R₆ is alkenyl group, both (Z) and (E)stereoisomeric forms of the compound are applicable.

For geometrical isomers of the compound in accordance with the presentinvention, methods for preparing geometrical isomers are also well knownin the art. For chiral isomers of the compound in accordance with thepresent invention, methods for preparing pure stereoisomers are wellknown in the art, such as synthesis with a chiral starting compound,chiral induction, enantioselective enzymatic conversions andchromatography of stereoisomers on chiral media.

The compounds of the formula I in accordance with the present inventioncan be in all isotopically labeled forms. For example, such compoundsare labeled with ₂H, ₃H, ₁₁C, ₁₃C, ₁₄C, ₁₃₁I, ₁₂₅I, ₁₂₃I and ₁₈F. Thelabeled compounds are for use in tracing a marker molecule in variousdiagnoses in vitro or in vivo.

In a preferred embodiment, the composition comprises the compound of theformula I or a pharmaceutically acceptable salt, solvate or stereoisomerthereof; and a carrier.

The carrier hereby refers to any substance that is pharmaceuticallyacceptable and, preferably, for assisting compound's in vivo absorptionand releasing in a sustained manner or other manners to modulate themetabolic kinetics of compound in a subject.

The term “pharmaceutically acceptable carrier” refers to various organicor inorganic carrier substances conventionally used as preparationmaterials. For example, excipient, disintegrant, binder, glidant,lubricant and the like for solid dosage forms, and for liquidpreparations, solvent, solubilizing agent, suspending agent, isotonicityagent, buffer, soothing agent and the like are used. If required,additives such as preservative, antioxidant, colorant, sweetening agentand the like are used.

In a preferred embodiment of the method for treating or alleviatingdiseases or disorders in accordance with the present invention, thediseases or disorders include, but not limited to, skin, lung, lymphnode, breast, cervix, uterus, gastrointestinal tract, ovary, prostate,colorectal, mouth, brain, head and neck, throat, testes, kidney,pancreas, bone, spleen, liver, bladder, larynx and nasal passage cancersor tumors.

Particularly, the cancers or tumors include, but not limited to,malignant gliomas and other sarcoma, carcinoma, adenoma and so on.

In a preferred embodiment, provided is a method for treating oralleviating malignant glioma, which has the following steps:administering the compound or a pharmaceutically acceptable salt,solvate or stereoisomer thereof in accordance with the present inventionto a subject suffering from malignant glioma.

In a preferred embodiment, the compound or a pharmaceutically acceptablesalt, solvate or stereoisomer thereof in accordance with the presentinvention can be combined with other pharmacologically active compoundin methods and compositions in accordance with the present invention.

In general, the compound of formula I in accordance with the presentinvention encompassing a series of 2-aryl-4-quinazolinones, which werenewly synthesized and exhibited significant anti-proliferative effectsto many kinds of human cancer cells. More specifically,2-aryl-4-quinazolinones display favorable in vivo and in vitro activityagainst malignant gliomas. 6-(Pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one, one of the 2-aryl-4-quinazolinones, displays excellentactivity against malignant gliomas and is an anti-malignant gliomacandidate.

In the following Examples, a series of novel 2-aryl-4-quinazolinoneshave been synthesized and displayed potent anti-cancer activity in vitrocancer cells and in vivo animal model. These cancer cells include humanmalignant melanoma (M21), human lung squamous carcinoma (CH27), humanhepatocellular carcinoma (Hep3B), lung non-small carcinoma (H460) cells,human oral squamous cell carcinoma (HSC-3), human colon adenocarcinoma(HT29-5FUR), human glioma cells (U87, U251, T98G, U373) and rat gliomacells (C6, RT2). Among these novel synthetic 2-aryl-4-quinazolinones,6-(pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one (compound 23,that is, MJ-66) is the most potent compound that showed significantcytotoxicity against a panel of human tumor cell lines with IC₅₀ valuesin the low micromolar to nanomolar concentration ranges. For example,compound 23 was shown being capable of inhibiting the growth of humanmalignant melanoma and glioma cells in vitro with IC₅₀ about 33 nM and60 nM respectively. In addition, compound 23 was demonstrated beingcapable of inhibiting tumor growth in glioma xenograft animal model.Therefore, the novel synthetic 2-aryl-4-quinazolinones and theirpharmaceutical compositions in accordance with the present invention arepotent for treating cancers. More specifically, 2-aryl-4-quinazolinonesdisplay favorable in vivo and in vitro activity against malignantgliomas and are suitable for treating human malignant gliomas.

EXAMPLES General Materials and Method

1. Materials and Reagents

All of the solvents and reagents were obtained commercially and usedwithout further purification. Reaction progress was monitored bythin-layer chromatography, using Merck TLC plates with fluorescentindicator. Column chromatography was performed on silica gel. Meltingpoints were determined with a Yanaco MP-500D melting point apparatus andare uncorrected. NMR spectra were obtained on a Bruker Avance DPX-200FT-NMR spectrometer in DMSO-d₆. The following abbreviations are used:mp, melting point, s, singlet; d, doublet; dd, double doublet; t,triplet; q, quartet; m, multiplet; and br, broad. MS spectra weremeasured with a Finnigan/Thermo Quest MAT 95XL instrument. Elementalanalyses (C, H, and N) were performed on a Perkin-Elmer 2400 Series IICHNS/O analyzer or Elementar vario EL III Heraeus CHNOS Rapid F002 andthe results were within ±0.4% of the calculated values.

MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium),MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide),N-acetylcysteine (NAC), cyclosporine A and dimethyl sulfoxide (DMSO)were purchased from SigmaeAldrich Corp. (St. Louis, Mo., USA). Fetalbovine serum (FBS), L-glutamine, penicillin-streptomycin, RPMI 1640medium, Dulbecco's modified eagle medium (DMEM), Minimum EssentialMedium (MEM) (Invitrogen), F12 medium, trypsin-EDTA, Neuralbasal Amedium (Gibco).

2. Cell Culture

M21 cell line (human malignant melanoma) was provided by Dr. Feng-YaoTang (China Medical University, Taiwan). Cells were grown in monolayerculture in RPMI-1640 medium, Eagle's minimum essential medium orDulbecco's modified Eagles medium (Invitrogen Corporation, Grand Island,N.Y., USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, Utah,USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco BRL,Rockville, Md., USA).

CH27 cells (Human lung squamous carcinoma) were grown in monolayerculture in Dulbecco's modified Eagle's medium (DMEM; Life Technologies,Rockville, Md., USA) containing 5% FBS (HyClone, Logan, Utah, USA), 100U/ml penicillin, 100 μml streptomycin (Gibco BRL, Rockville, Md., USA)and 2 mM glutamine (Merck, Darmstadt, Germany).

Hep3B (human hepatocellular carcinoma) and H460 (lung non-smallcarcinoma) cells were grown in monolayer culture in Dulbecco's modifiedEagle's medium (DMEM) (Life Technologies, Rockville, Md., USA)containing 5% fetal bovine serum (HyClone, Logan, Utah, USA), 100 U/mlpenicillin and 100 μg/ml streptomycin (Gibco BRL, Rockville, Md., USA)and 2 mM glutamine (Merck, Darmstadt, Germany).

The HSC-3 (human oral squamous cell carcinoma) were cultured in 1:1Dulbecco's Modified Eagle's Medium (DMEM) and Ham's Nutrient MixtureF-12, containing 10% FBS (HyClone, Logan, Utah, USA), 100 U/mlpenicillin, 100 μg/ml streptomycin (Gibco BRL, Rockville, Md., USA) and2 mM glutamine (Merck, Darmstadt, Germany).

HT29-5FUR cell line (human colon adenocarcinoma) provided by Dr.Jai-Sing Yang (China Medical University, Taiwan) was cultured inRPMI-1640 medium (Invitrogen, Carlsbad, Calif., USA) supplemented with10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin.

The human glioma cell lines U87, U251, T98G, U373, and rat glioma cellline RT2 provided by Dr. Michael Hsiao (Genomics Research Center,Academia Sinica, Taiwan) were cultured in Dulbecco's Modified Eaglemedium (DMEM) (Caisson) supplemented with 10% fetal bovine serum (FBS)(Sigma-Aldrich), 2 mM L-glutamine (Caisson), 100 U/ml penicillin, and100 μg/ml streptomycin (Caisson). The rat glioma C6 cell line providedby Dr. Shun-Fen Tzeng (National Cheng Kung University, Taiwan) wascultured in DMEM/F12 (Caisson) supplemented with 10% fetal bovine serum,2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Thehuman normal glia cell line SVGP12, kindly provided by Dr. MichaelHsiao, was cultured in Minimum Essential Medium (MEM) (Invitrogen)supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mlpenicillin, and 100 μg/ml. Primary glia cells were prepared by surgery.Cerebral cortices without meninges were dissected from postnatal (P₀-P₂)Sprague-Dawley (SD, NCKU Laboratory Animal Center) rats and dissociatedin 0.05% trypsin-EDTA (Biowest) at 37° C. for 10 min. Culture medium wasadded and pipetted for several times, and supernatant was removed aftera centrifugation at 1,000 rpm for 5 minutes. Glia cells were filteredthrough 70 μm cell strainer (BD Falcon) and then cultured in NeuralbasalA medium (Gibco) supplemented with 5% fetal bovine serum(Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/mlstreptomycin and 17.5 mM D-glucose on 50 μg/ml poly-D-lysine(Sigma-Aldrich) pre-coated dish.

All cells were maintained in a humidified incubator at 37° C. and 5%CO₂/95% air.

3. Cell Proliferation and Viability Assay

3.1 MTS Assay

Cell proliferation was determined by MTS (tetrazolium compound,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay (Promega). MTS assay is a colorimetric assay for determining cellviability (Mosmann, T. (1983). Rapid colorimetric assay for cellulargrowth and survival: application to proliferation and cytotoxicityassays. J Immunol Methods 65, 55-63). The MTS tetrazolium compound isbio-reduced by NADPH or NADH produced by dehydrogenase in live cellsinto a colored formazan product. 1×10³ Cells were seeded in each well of96-well plates for MTS assay and cells were incubated for 24 hr. Culturemedia containing sequential concentrations of MJ-66, MJ-68 and MJ-78 (1,0.1, 0.05, 0.025, 0.0125 and 0.00625 μM) or DMSO (0.1%) were added toeach well, and cells were incubated at 37° C. At the indicated timepoints (0, 24, 48 and 72 hr), media were removed, and then fresh culturemedia (100 μl/well) with MTS solution (20 μl/well) were added, and cellswere incubated at 37° C. for 1-4 hr. The absorbance of soluble formazanwas measured at 490 nm with microplate reader (Molecular device). Thecell viability was determined by the percentage of the absorptionrelative to untreated control. All results were performed from threeindependent experiments.

3.2 MTT Assay

Cells (1×10⁴ cells/well) in 96-well plates were exposed to test MJ-66,and 0.1% DMSO in media served as a vehicle control. After 24 and 48hr-incubations, 100 ml MTT (0.5 mg/mL) solution was added to each well,the plate was incubated at 37° C. for 4 hours. Then, 100 ml 0.04 N HClin isopropanol was added and the absorbance at 570 nm was measured foreach well. The cell survival rate was expressed as % of control. Allresults were performed from three independent experiments.

3.3 Trypan-Blue Exclusion Assay

Cell viability was also determined by trypan-blue exclusion assay. Cellswere seeded in 6-well plates (2×10⁴/well) for trypan blue exclusionassay, and incubated for 24 hours at 37° C. Culture medium containingtest compound or DMSO (0.006%) was added to each well, and cells wereincubated at 37° C. At indicated time points (0, 24, 48, 72 and 96 hr),cells were suspended with 0.05% trypsin-EDTA, and stained withtrypan-blue dye (0.4%) (Sigma). The cell viability was evaluated by thepercentage of death relative to the total cell, and cell growth curvewas determined by number of live cells relative to the total number ofcells.

4. Cell Cycle Assay

To analyze cellular DNA content by flow cytometry, 3×10⁵ cells wereseeded in 10 cm dish and incubated for 24 hours at 37° C. Culture mediumcontaining MJ-66 (IC₅₀) or DMSO (0.006%) was added, and cells wereincubated for 24 hours at 37° C. At indicated time points (0, 6, 12, 24and 48 hr), over 10⁶ cells were suspended with 0.05% trypsin-EDTA, andfixed in 70% ethanol over 1 hour at −20° C. After washed byphosphate-buffered saline (PBS) twice, cells were then suspended with 1ml propidium iodide/Triton-X 100 staining solution (20 μg/ml PI, 0.1%Triton-X 100 and 0.2 mg/ml ribonuclease A (RNase A, Sigma) in PBS) andincubated for 30 minutes at room temperature in the dark. After filteredby 35 μm nylon mesh (Falcon, 352235), the DNA content was then analyzedby flow cytometry (FACS can, BD Bioscience). The cell-cycle distribution(subG1, G1, S, G2/M, and >4N DNA content) of ten thousand cells wasanalyzed by WinMDI software.

5. G1 Synchronized by Ara-C

To analyze the effect of cytosine arabinoside (Ara-C, a DNA synthesisinhibitor) on the MJ-66-treated glioma cells by flow cytometry, afterseeding 3×10⁵ cells in 10 cm-plate for at least 24 hours at 37° C.,glioma cells were then treated with Ara-C (4.17 mM/ml) for 12 hours, andthen MJ-66 (IC₅₀) or DMSO (0.006%) was added. At indicated time points(0, 6, 12 and 24 hr), >10⁶ cells were collected and fixed in 70% ethanolover 1 hour at −20° C. After staining with PI as described, DNA contentof ten thousand cells was analyzed by flow cytometry.

6. Immunofluorescent Staining

2×10⁴ cells were seeded on the PDL-coating 12 mm-glass coverslips in24-well plate and allowed to attach for 24 hours at 37° C. Culturemedium containing MJ-66 (IC₅₀) or DMSO (0.006%) was added, and cellswere incubated at 37° C. At indicated time points (0, 12 and 24 hr),cells were fixed in 4% paraformaldehyde (PFA) in PBS for 30 minutes.After permeabilized by 0.2% Triton X-100 in 0.1 M PBS for 10 minutes and10% methanol containing 0.2% Triton X-100 in 0.1 M PBS for 5 minutes,cells were blocked in 3% bovine serum albumin (BSA, Sigma) for 1 hour.The cells were immunostained for mitotic spindle with mouse monoclonalalpha-tubulin (Sigma-Aldrich), and mitotic center with rabbit polyclonalgamma-tubulin (Genetex), and then stained with appropriated secondaryantibodies conjugated with Texas Red or FITC for 1 hr. Nuclei werestained with Hoechst 33342 for 10 minutes (0.5 μg/ml, Sigma-Aldrich,B2261). Fluorescence images were detected by confocal laser scanningmicroscope (FV1000, Olympus).

7. In Vivo Xenograft Animal Model

For tumorigenesis, U87 glioma cells (1×10⁶ cells per 100 μl PBS) wereinoculated subcutaneously into the right flank of 8-to-10-week-old malenude mice (BALB/cAnN-Foxnlnu/CrlNarl mice, National Laboratory AnimalCenter, Taiwan). Tumor growth was measured every three days. Tumorvolume was calculated by volume (mm³)=(length×width)/2 (Zhou, Y. H., Wu,X., Tan, F., Shi, Y. X., Glass, T., Liu, T. J., Wathen, K., Hess, K. R.,Gumin, J, Lang, F., et al. (2005). PAX6 suppresses growth of humanglioblastoma cells. J Neurooncol 71, 223-229). When the tumors reached amean volume of 50 to 70 mm³, MJ-66 (1.36 microgram/kg in saline) orvehicle (DMSO or saline) was injected intratumor once per two daysduring 20 days.

8. Statistical Analysis

Experiments were performed at least in triplicate. All results wereexpressed as the mean±standard error of the mean. Independentexperiments were analyzed by unpaired t test. Levels of P<0.05 wereconsidered to be of statistical significance.

Preparation Example 1 General Procedure for Synthesis of Starting5-substituted-2-aminobenzamides (9-13) by Schemes 1 and 2

Starting 2-nitrobenzamide for synthesis of 2-aryl-4-quinazolinone offormula I in accordance with the present invention was prepared asfollows.

5-Substituted-2-nitrobenzamide (2, 5-8) was prepared using standardmethodology from 5-substituted-2-nitrobenzoic acid (1, 3) by reactionwith reagents, such as SOCl₂, NH₃, and dimethylamine (or pyrrolidine,piperidine, morpholine), under a condition as shown in the generalreaction sequence in Schemes 1 and 2. Compounds 2 and 5-8 were treatedwith H₂/Pd—C in methanol to afford 5-substituted-2-aminobenzamide(9-13).

The procedure for synthesis of 5-substituted-2-aminobenzamides wasperformed as follows:

(a) (i) Thionyl chloride (1.5 g, 12.6 mmol) was added dropwise to asuspension of a 5-substituted-2-nitrobenzoic acid (1: 1.2 g, 6.3 mmol;3: 1.3 g, 6.3 mmol) in dichloroethane (30 mL) under reflux. Theresulting mixture was stirred under reflux for 4 hours and dried undervacuum. (ii) The residue was dissolved in 200 mL of dichloroethane andtreated with anhydrous ammonia gas at room temperature. After removingsolvent, the intermediate 5-methoxy-2-nitrobenzamide (2: 1.1 g, 92%) or5-chloro-2-nitrobenzamide (4: 1.2 g, 92%) was obtained.

(b) The intermediate 4 was dissolved in DMF and reacted withN,N-dimethylamine (or pyrrolidine or piperidine or morpholine) at 110°C. to afford 5-substituted-2-nitrobenzamides (5: 1.1 g, 88%; 6: 1.2 g,86%; 7: 1.2 g, 80%; 8: 1.2 g, 81%). (c) The5-substituted-2-nitrobenzamides (2, 5-8) was dissolved in MeOH andhydrogenated over 10% Pd/C for 1 to 2 hours. The catalyst was removed byfiltration, and the solution was dried under vacuum to afford the5-substituted-2-aminobenzamides 9-13 (9: 0.8 g, 86%; 10: 0.9 g, 85%; 11:0.9 g, 85%; 12: 0.9 g, 82%; 13: 0.9 g, 82%) as brown powders.

Preparation Example 2 General Procedure for Synthesis of6-substituted-2-aryl-4-quinazolinones

6-substituted-2-aryl-4-quinazolinones were derived from Compounds 9-13obtained in Preparation example 1 in this example. First, compounds 9-13then were respectively reacted with substituted benzaldehydes inN,N-dimethylacetamide (DMAC) in the presence of NaHSO₃ at 150° C. Then,Thermal cyclodehydration/dehydrogenation gave the2,6-disubstituted-4-quinazolinones (17-31) as shown in scheme 3.

The procedure for synthesis of 6-substituted-2-aryl-4-quinazolinones wasperformed as follows:

Sodium hydrogen sulfite (0.8 g, 7.5 mmol) was added to a solution of5-methoxy-2-aminobenzamide (9) (1.2 g, 7.3 mmol) and 1-naphthaldehyde(14) (1.1 g, 7.3 mmol) in N,N-dimethylacetamide (DMAC) (20 mL). Themixture was heated with stirring at 150° C. for 3 h and poured into icewater (200 mL). The precipitate was collected, washed with water, anddried in vacuo. After purification by column chromatography (silica gel;chloroform) and then recrystallization from EtOH,6-methoxy-2-(naphthalen-1-yl)-4-quinazolinone (17) was obtained (1.5 g,70%) as pale yellow needles. The method used to prepare 17 was used withthe indicated substituted benzaldehyde and benzamide to afford 18-31.Compounds 17-31 were further analyzed by the method as described in“General materials and method”. The properties of Compounds 17-31 wererespectively described as below.

Example 1 6-Methoxy-2-(naphthalen-1-yl) quinazolin-4-one (17)

Compound 17 was synthesized from 5-methoxy-2-aminobenzamide (9) (1.2 g,7.3 mmol) and 1-naphthaldehyde (14) (1.1 g, 7.3 mmol): yield 1.5 g(70%); pale yellow needles; mp 174 to 175° C.; ¹H NMR (DMSO-d₆) δ 3.87(3H, s, OCH₃), 7.22-7.34 (2H, m, 2×ArH), 7.53-7.72 (5H, m, 5×ArH),7.95-8.22 (3H, m, 3×ArH), 12.62 (1H, br s, NH) ppm; MS (ESI) m/z 302;Anal. (C₁₉H₁₄N₂O₂): C, H, N.

Example 2 6-Methoxy-2-(1H-indol-3-yl) quinazolin-4-one (18)

Compound 18 was synthesized from 5-methoxy-2-aminobenzamide (9) (1.2 g,7.3 mmol) and indo-3-carboxaldehyde (15) (1.1 g, 7.3 mmol): yield 1.4 g(68%); brown crystals; mp 311 to 312° C.; ¹H NMR (DMSO-d₆) δ 3.84 (3H,s, OCH₃), 7.15-7.19 (2H, m, 2×ArH), 7.35-7.49 (3H, m, 3×ArH), 7.63-7.68(1H, m, 1×ArH), 8.45 (1H, s, 1×ArH), 8.46-8.63 (1H, m, 1×ArH), 11.76(1H, s, NH), 12.11 (1H, br s, NH) ppm; MS (ESI) m/z 291; Anal.(C₁₇H₁₃N₃O₂): C, H, N.

Example 3 6-Methoxy-2-(benzo[b]thiophen-3-yl) quinazolin-4-one (19)

Compound 19 was synthesized from 5-methoxy-2-aminobenzamide (9) (1.2 g,7.3 mmol) and thianaphthene-3-carboxaldehyde (16) (1.2 g, 7.3 mmol):yield 1.3 g (60%); brown crystals; mp 289 to 290° C.; ¹H NMR (DMSO-d₆) δ3.89 (3H, s, OCH₃), 7.43-7.57 (4H, m, 4×ArH), 7.76-7.80 (1H, m, 1×ArH),8.07-8.11 (1H, m, 1×ArH), 8.73 (1H, s, 1×ArH), 8.98-9.01 (1H, m, 1×ArH),12.49 (1H, br s, NH) ppm; MS (ESI) m/z 308; Anal. (C₁₇H₁₂N₂O₂S): C, H,N.

Example 4 6-(N,N-Dimethylamino)-2-(naphthalen-1-yl) quinazolin-4-one(20)

Compound 20 was synthesized from 5-N,N-dimethyl-2-aminobenzamide (10)(1.3 g, 7.3 mmol) and 1-naphthaldehyde (14) (1.1 g, 7.3 mmol): yield 1.5g (65%); yellow crystals; mp 254 to 255° C.; ¹H NMR (DMSO-d₆) δ 2.99(6H, s, N(CH₃)₂), 7.22-7.34 (2H, m, 2×ArH), 7.51-7.72 (5H, m, 5×ArH),7.96-8.23 (3H, m, 3×ArH), 12.39 (1H, br s, NH) ppm; MS (ESI) m/z 315;Anal. (C₂₀H₁₇N₃O): C, H, N.

Example 5 6-(N,N-Dimethylamino)-2-(1H-indol-3-yl)quinazolin-4-one (21)

Compound 21 was synthesized from 5-N,N-dimethyl-2-aminobenzamide (10)(1.3 g, 7.3 mmol) and indo-3-carboxaldehyde (15) (1.1 g, 7.3 mmol):yield 1.3 g (60%); yellow crystals; mp>300° C.; ¹H NMR (DMSO-d₆) δ 2.99(6H, s, N(CH₃)₂)_(,) 7.17-7.34 (4H, m, 4×ArH), 7.43-7.47 (1H, m, 1×ArH),7.58-7.63 (1H, m, 1×ArH), 8.44 (1H, s, 1×ArH), 8.65-8.69 (1H, m, 1×ArH),11.71 (1H, s, NH), 11.92 (1H, br s, NH) ppm; MS (ESI) m/z 304; Anal.(C₁₈H₁₆N₄O): C, H, N.

Example 6 6-(N,N-Dimethylamino)-2-(benzo[1)]thiophen-3-yl)quinazolin-4-one (22)

Compound 22 was synthesized from 5-N,N-dimethyl-2-aminobenzamide (10)(1.3 g, 7.3 mmol) and thianaphthene-3-carboxaldehyde (16) (1.2 g, 7.3mmol): yield 1.5 g (62%); pale yellow needles; mp>300° C.; ¹H NMR(DMSO-d₆) δ 2.99 (6H, s, N(CH₃)₂), 7.20-7.67 (5H, m, 5×ArH), 8.02-8.05(1H, m, 1×ArH), 8.63 (1H, s, 1×ArH), 8.97-9.01 (1H, m, 1×ArH), 12.21(1H, br s, NH) ppm; MS (ESI) m/z 321; Anal. (C₁₈H₁₅N₃OS): C, H, N.

Example 7 6-(Pyrrolidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one (23)(MJ-66)

Compound 23 was synthesized from 5-pyrrolidinyl-2-aminobenzamide (11)(1.5 g, 7.3 mmol) and 1-naphthaldehyde (14) (1.1 g, 7.3 mmol): yield 1.6g (65%); pale yellow needles; mp 276 to 277° C.; ¹H NMR (DMSO-d₆) δ 1.99(4H, m, CH ₂CH₂NCH₂CH ₂), 3.27 (4H, m, CH₂NCH₂), 7.10-7.21 (2H, m,2×ArH), 7.56-7.76 (5H, m, 5×ArH), 8.01-8.21 (3H, m, 3×ArH), 12.34 (1H,br s, NH) ppm; MS (ESI) m/z 341; Anal. (C₂₂H₁₉N₃O): C, H, N.

Example 8 6-(Pyrrolidin-1-yl)-2-(1H-indol-3-yl)quinazolin-4-one (24)

Compound 24 was synthesized from 5-pyrrolidinyl-2-aminobenzamide (11)(1.5 g, 7.3 mmol) and indo-3-carboxaldehyde (15) (1.1 g, 7.3 mmol):yield 1.5 g (61%); brown powders; mp 298° C.; ¹H NMR (DMSO-d₆) δ 1.94(4H, m, CH ₂CH₂NCH₂CH ₂), 3.28 (4H, m, CH₂NCH₂), 6.97-7.20 (4H, m,4×ArH), 7.39-7.43 (1H, m, 1×ArH), 7.53-7.58 (1H, m, 1×ArH), 8.38 (1H, s,1×ArH), 8.61-8.65 (1H, m, 1×ArH), 11.68 (1H, s, NH), 11.88 (1H, br s,NH) ppm; MS (ESI) m/z 330; Anal. (C₂₀H₁₈N₄O): C, H, N.

Example 9 6-(Pyrrolidin-1-yl)-2-(benzo[b]thiophen-3-yl)quinazolin-4-one(25) (MJ-78)

Compound 25 was synthesized from 5-pyrrolidinyl-2-aminobenzamide (11)(1.5 g, 7.3 mmol) and thianaphthene-3-carboxaldehyde (16) (1.2 g, 7.3mmol): yield 1.5 g (60%); brown powders; mp>300° C.; ¹H NMR (DMSO-d₆) δ1.97 (4H, m, CH ₂CH₂NCH₂CH ₂), 3.47 (4H, m, CH₂NCH₂), 7.43-7.57 (4H, m,4×ArH), 7.76-7.80 (1H, m, 1×ArH), 8.07-8.11 (1H, m, 1×ArH), 8.73 (1H, s,1×ArH), 8.98-9.02 (1H, m, 1×ArH), 12.51 (1H, br s, NH) ppm; MS (ESI) m/z347; Anal. (C₂₀H₁₇N₃OS): C, H, N.

Example 10 6-(Piperidin-1-yl)-2-(naphthalen-1-yl)quinazolin-4-one (26)(MJ-68)

Compound 26 was synthesized from 5-piperidinyl-2-aminobenzamide (12)(1.6 g, 7.3 mmol) and 1-naphthaldehyde (14) (1.1 g, 7.3 mmol): yield 1.6g (65%); brown needles; mp 233 to 235° C.; ¹H NMR (DMSO-d₆) δ 1.57-1.62(6H, m, (CH ₂)₂CH₂NCH₂CH ₂), 3.26-3.31 (4H, m, CH₂NCH₂), 7.48-7.65 (6H,m, 6×ArH), 7.72-7.76 (1H, m, 1×ArH), 8.00-8.13 (3H, m, 3×ArH), 12.43(1H, br s, NH) ppm; MS (ESI) m/z 355; Anal. (C₂₃H_(2i)N₃O): C, H, N.

Example 11 6-(Piperidin-1-yl)-2-(1H-indol-3-yl)quinazolin-4-one (27)

Compound 27 was synthesized from 5-piperidinyl-2-aminobenzamide (12)(1.6 g, 7.3 mmol) and indo-3-carboxaldehyde (15) (1.1 g, 7.3 mmol):yield 1.6 g (64%); brown crystals; mp>300° C.; ¹H NMR (DMSO-d₆) δ1.59-1.62 (6H, m, (CH ₂)₂CH₂NCH₂CH ₂), 3.20-3.24 (4H, m, CH₂NCH₂),7.18-7.21 (2H, m, 2×ArH), 7.41-7.48 (3H, m, 3×ArH), 7.50-7.62 (1H, m,1×ArH), 8.46 (1H, s, 1×ArH), 8.65-8.69 (1H, m, 1×ArH), 11.73 (1H, s,NH), 11.96 (1H, br s, NH) ppm; MS (ESI) m/z 344; Anal. (C₂₁H₂₀N₄O): C,H, N.

Example 12 6-(Piperidin-1-yl)-2-(benzo[1)]thiophen-3-yl)quinazolin-4-one(28)

Compound 28 was synthesized from 5-piperidinyl-2-aminobenzamide (12)(1.6 g, 7.3 mmol) and thianaphthene-3-carboxaldehyde (16) (1.2 g, 7.3mmol): yield 1.6 g (60%); pale yellow crystals; mp>300° C.; ¹H NMR(DMSO-d₆) δ 1.57-1.59 (6H, m, (CH ₂)₂CH₂NCH₂CH ₂), 3.22-3.25 (4H, m,CH₂NCH₂), 7.42-7.54 (4H, m, 4×ArH), 7.63-7.67 (1H, m, 1×ArH), 8.03-8.07(1H, m, 1×ArH), 8.66 (1H, s, 1×ArH), 8.97-9.00 (1H, m, 1×ArH), 12.30(1H, br s, NH) ppm; MS (ESI) m/z 361; Anal. (C₂₁H₁₉N₃OS): C, H, N.

Example 13 6-(Morpholino)-2-(naphthalen-1-yl)quinazolin-4-one (29)

Compound 29 was synthesized from 5-morpholinyl-2-aminobenzamide (13)(1.6 g, 7.3 mmol) and 1-naphthaldehyde (14) (1.1 g, 7.3 mmol): yield 1.7g (64%); yellow crystals; mp 291 to 293° C.; ¹H NMR (DMSO-d₆) δ 3.22(4H, t, J=4.7 Hz, CH₂NCH₂), 3.77 (4H, t, J=4.7 Hz, CH₂OCH₂), 7.51-7.66(6H, m, 6×ArH), 7.73-7.76 (1H, m, 1×ArH), 8.00-8.11 (3H, m, 3×ArH),12.48 (1H, br s, NH) ppm; MS (ESI) m/z 357; Anal. (C₂₂H₁₉N₃O₂): C, H, N.

Example 14 6-(Morpholino)-2-(1H-indol-3-yl)quinazolin-4-one (30)

Compound 30 was synthesized from 5-morpholinyl-2-aminobenzamide (13)(1.6 g, 7.3 mmol) and indo-3-carboxaldehyde (15) (1.1 g, 7.3 mmol):yield 1.6 g (65%); brown crystals; mp>300° C.; ¹H NMR (DMSO-d₆) δ 2.97(4H, t, J=4.7 Hz, CH₂NCH₂), 3.74 (4H, t, J=4.7 Hz, CH₂OCH₂), 7.12-7.22(2H, m, 2×ArH), 7.41-7.63 (4H, m, 4×ArH), 8.43 (1H, s, 1×ArH), 8.60-8.64(1H, m, 1×ArH), 11.76 (1H, s, NH), 12.01 (1H, br s, NH) ppm; MS (ESI)m/z 346; Anal. (C₂₀H₁₈N₄O₂): C, H, N.

Example 15 6-(Morpholino)-2-(benzo[b]thiophen-3-yl) quinazolin-4-one(31)

Compound 31 was synthesized from 5-morpholinyl-2-aminobenzamide (13)(1.6 g, 7.3 mmol) and thianaphthene-3-carboxaldehyde (16) (1.2 g, 7.3mmol): yield 1.6 g (60%); brown crystals; mp>300° C.; ¹H NMR (DMSO-d₆) δ2.47 (4H, t, J=4.7 Hz, CH₂NCH₂), 3.75 (4H, t, J=4.7 Hz, CH₂OCH₂),7.44-7.59 (4H, m, 4×ArH), 7.67-7.72 (1H, m, 1×ArH), 8.03-8.07 (1H, m,1×ArH), 8.67 (1H, s, 1×ArH), 8.96-9.00 (1H, m, 1×ArH), 12.31 (1H, br s,NH) ppm; MS (ESI) m/z 363; Anal. (C₂₀H₁₇N₃O₂S): C, H, N.

Example 16 In Vitro Cytotoxicity of 2-Aryl-4-quinazolinones

2-Aryl-4-quinazolinones prepared in Examples 1 to 15, Compounds 17-31,were listed in the following Table 1, denoted as MJ-66 to −80 andidentified by their molecular weight.

TABLE 1 Item Molecular (Compound) Structure Formula weight MJ-65 (24)

C₂₀H₁₈N₄O 330 MJ-66 (23)

C₂₂H₁₉N₃O 341 MJ-67 (29)

C₂₂H₁₉N₃O₂ 357 MJ-68 (26)

C₂₃H₂₁N₃O 355 MJ-69 (20)

C₂₀H₁₇N₃O 315 MJ-70 (17)

C₁₉H₁₄N₂O₂ 302 MJ-72 (27)

C₂₁H₂₀N₄O 344 MJ-73 (28)

C₂₁H₁₉N₃OS 361 MJ-74 (18)

C₁₇H₁₃N₃O₂ 291 MJ-75 (21)

C₁₈H₁₆N₄O 304 MJ-76 (30)

C₂₀H₁₈N₄O₂ 346 MJ-77 (31)

C₂₀H₁₇N₃O₂S 363 MJ-78 (25)

C₂₀H₁₇N₃OS 347 MJ-79 (19)

C₁₇H₁₂N₂O₂S 308 MJ-80 (22)

C₁₈H₁₅N₃OS 321

The anti-proliferation effects of Compounds 17-31 on M21, CH27, H460,Hep3B and HSC-3 cells were evaluated by the trypan blue exclusion assay.The results, IC₅₀ values of Compounds 17-31 on cancer cell lines, arepresented in Table 2. The results demonstrated that the Compounds 17,20, 23, 24, 26, 27 and 29 exhibited cytotoxic effects toward thesecancer cells, but compound 17 was inactive on HSC-3 cells. Among them,Compound 23, that is, MJ-66, was the most potent compound against thesehuman cancer cells in vitro.

TABLE 2 In vitro cytotoxicity of 2-Aryl-4-quinazolinones IC₅₀ (μM)*Compound M21 CH27 H460 Hep3B HSC-3 17 6.093 0.089 0.079 1.870 >1018 >10 >10 >10 >10 >10 19 >10 >10 >10 >10 >10 20 3.775 0.447 0.506 8.6260.202 21 >10 >10 >10 >10 >10 22 >10 >10 >10 >10 >10 23 0.033 0.053 0.0751.350 0.041 24 7.629 8.700 8.810 7.390 1.890 25 >10 >10 >10 >10 >10 261.587 0.362 0.376 7.235 1.730 27 8.989 >10 6.862 5.653 >1028 >10 >10 >10 >10 >10 29 2.333 0.597 0.432 1.056 0.61430 >10 >10 >10 >10 >10 31 >10 >10 >10 >10 >10 *IC₅₀: half maximalinhibitory concentration; M21: skin cancer, CH27: human lung squamouscarcinoma, H460: human non-small cell lung cancer, Hep3B: humanhepatoma, HSC-3: human squamous carcinoma; Cell viability was determinedby trypan blue exclusion assay.

Example 17 2-Aryl-4-quinazolinone (MJ-66) inhibited HT29-5FUR cells(Human Colon Adenocarcinoma) Proliferation

The growth inhibition effects of MJ-66 on HT29-5FUR cells wereinvestigated. For cell viability assay, HT29-5FUR cells were culturedand treated with 0, 25, 50, 100 and 200 nM MJ-66 and the percentages ofviable cells were detected by MTT assay described in “General materialsand methods”.

As shown in FIG. 1, exposure to various concentrations of MJ-66 (25, 50,100 and 200 nM) for 24 and 48 hours (denoted as 24 h and 48 h) resultedin dose-dependent decreases on cell number of HT29-5FUR relative tocontrol cultures.

Example 18 2-Aryl-4-quinazolinones Inhibited Glioma Cells Proliferation

To investigate the effect of 2-aryl-4-quinazolinone in accordance withthe present invention on cell proliferation, glioma cell lines weretreated with MJ-66, MJ-68, or MJ-78. Structures of MJ-66, MJ-68 andMJ-78 were shown in Table 1. To determine the effects among MJ-66,MJ-68, and MJ-78 on C6 rat glioma (denoted as C6) and U87 human gliomacell lines (denoted as U87), cells were treated with differentconcentrations of the 2-aryl-4-quinazolinone (0.00625, 0.0125, 0.025,0.05, 0.1 and 1 μM) for 48 hours. The cell viability of each group wasanalyzed by MTS assay (mean±SEM, n=3). As shown in FIG. 2B, survivalrates of glioma cells were effectively inhibited by MJ-66 at the 48thhr. (IC₅₀ values MJ-66, C6=0.056±0.009 μM, U87=0.054±0.01 μM). However,MJ-68 or MJ-78 did not inhibit glioma cell survival significantly asMJ-66 (IC₅₀: MJ-68, C6=0.47±0.16 μM, U87=0.54±0.24 μM; MJ-78, C6>1 μM,U87>1 μM). These results indicated that MJ-66 had significantcytotoxicity to glioma cells.

The effects of MJ-66 on other glioma cell lines were further examined.Rat glioma cell lines including C6 and RT2, and human glioma cell linesincluding U87, U251, U373 and T98G, were treated with serialconcentrations (0.00625, 0.0125, 0.025, 0.05, 0.1 and 1 μM) of MJ-66 atthe 24th, 48th and 72nd hours (denoted as 24, 48 and 72 hr). Cellviability was assessed by MTS assay (mean±SEM, n=3). Glioma cellsurvival was treated with MJ-66 for various time periods.

As shown in FIGS. 3A and 3B, the survival rates of MJ-66-treated gliomacell lines were decreased in a time- and concentration-dependent manner,whereas 0.1% DMSO had no effect on glioma cell viability.

Example 19 2-Aryl-4-quinazolinone (MJ-66) Induced Glioma Cell Death byTrypan Blue Exclusion Assay

To investigate the glioma cell death induced by MJ-66, cell viabilitywas assessed by trypan blue exclusion assay. C6 and U87 glioma cellstreated with MJ-66 (30, 60, 90 nM) or DMSO (0.009%) for 0, 24, 48, 72and 96 hours (hr) were observed under bright field microscope (FIG. 4A).The percentages of cell death and cell growth curve were determined bytrypan blue assay followed by cell counting (mean±SEM, n=3). Relative tototal cell number, trypan blue positive cells were defined as deadcells.

As shown in FIG. 4A, the morphology of MJ-66-treated glioma cells (upperpanel: C6 and lower panel: U87) was rounding up, and the ability toattach also decreased in a time- and concentration-dependent manner. Asshown in FIG. 4B, the percentage of dead cells of MJ-66 treated cellswas significantly induced at the 72nd hour in C6 glioma cells (upperleft and lower left panels) and the 48th hour in U87 glioma cells (upperright and lower right panels)(*p<0.05 versus control).

Example 20 2-Aryl-4-quinazolinone (MJ-66) Induced Glioma G2/M Arrest andCell Death

To investigate the cell cycle distribution of MJ-66-treated glioma cell,C6 glioma cells were treated with 60 nM MJ-66 for 0, 6, 12, 24, 36 and48 hours, and the cell cycle distributions of the percentages of cellsat subG1, G1, S and G2/M phases and DNA content of cells were monitoredby flow cytometry with propidium iodide staining.

As shown in FIG. 5A, flow cytometry showed that MJ-66 treatment of C6glioma cells significantly increased G2/M cell population after 6 and 12hours. Further, MJ-66 increased sub G1 and >4N DNA content cellpopulation after 24, 36 and 48 hours in a time-dependent manner (FIG.5B). These data suggest that MJ-66 effectively induced glioma G2/Marrest, then undergoing cell death. However, whether MJ-66 hascytotoxicity to normal glia cells was not determined. In the nextExample the effects of MJ-66 in normal glia cells were further examined.

Example 21 2-Aryl-4-quinazolinone (MJ-66) did not CauseNon-Proliferating Glioma Cell to Undergo Cell Death

To investigate the influence of MJ-66 on normal glia such as human gliacell line SVGP12 and rat primary glia cells, cells were treated withdifferent concentrations of MJ-66 in vitro (0.00625, 0.0125, 0.025,0.05, 0.1 and 1 μM) for different time durations (0, 6, 12 and 24 hours)and the cell viability was assessed by MTS assay. Cells alone or treatedwith DMSO were used as control. As shown in FIG. 6A, cell viability ofSVGP12 and rat glia cells was inhibited by MJ-66. We suggest that gliaproliferate constantly in vitro, so that the MJ-66 in vitro also hascytotoxicity to normal glia. However, most glia cells in animals arestatic and their proliferation rate is almost silent. Therefore,cytosine arabinoside (Ara-C), a DNA synthesis inhibitor, was furtherused to inhibit glioma cell proliferation and glioma cells were thentreated with MJ-66 or DMSO as described in “General materials andmethods”. Specifically, after C6 glioma cells were treated with 41.6 nMAra-C for 12 hours, and then treated with solvent (DMSO) or 60 nM MJ-66for 0, 6, 12 and 24 hours, the cell cycle distributions and DNA contentof the cells were monitored by flow cytometry with propidium iodidestaining C6 glioma cells treated with 41.6 nM Ara-C for 12 hours (12 hr)were detected by flow cytometry with propidium iodide staining ascontrol.

As shown in FIG. 6B, C6 glioma cells treated with Ara-C for 12 hourswere G1 arrest. Compared to cells without treatment with Ara-C, G1arrest glioma cells were not induced cell death by MJ-66 at 12th and24th hours (FIG. 6C). These results indicated that MJ-66 causedproliferating glioma cell death, but did not induce non-proliferatingglioma cell undergoing cell death.

Example 22 2-Aryl-4-quinazolinone (MJ-66) Induced Glioma Cell Apoptosis

To observe the morphology of MJ-66-treated glioma cells, C6 or U87glioma cells were treated with medium or 60 nM MJ-66 for 24 hours andstained with Hoechst 33342 and antibodies against alpha-tubulinconjugated with FITC or antibodies against gamma-tubulin conjugated withFITC in combination with antibodies against alpha-tubulin conjugatedwith Texas Red were detected by confocal laser scanning microscope.Fluorescence images revealed that U87 glioma cells treated with MJ-66exhibited abnormal nuclear phenotype (FIGS. 7A and 7B), while controlcells did not. MJ-66-treated cells possessed multinuclear giant cells.In the early stage, MJ-66-treated cells were observed to have aberrantmultipolar spindle and unaligned chromosomes, which were quantified asshown in FIG. 7C. The formation of multinuclear and multipolar spindleis typical characteristic of mitotic catastrophe. Mitotic catastrophe isdefined as a type of cell death due to abnormal mitosis. The resultsdemonstrated that MJ-66 induced cell death of glioma cells.

Example 23 2-Aryl-4-quinazolinone (MJ-66) Inhibited Tumor Growth inXenograft Animal Model

Since MJ-66 was able to effectively cause glioma to undergo mitoticcatastrophe in vitro, whether MJ-66 could inhibit tumor growth in vivowas further examined by U87 human glioma xenograft animal model.Approximately 1×10⁶ U87 glioma cells were injected subcutaneously intothe nude mice and when tumors size reached 50 to 200 mm³ in volume,MJ-66 (1.36 microgram/kg in saline) or vehicle (DMSO or saline denotedrespectively as DMSO and Control) was administered by intra-tumor every2 days for 10 times and tumor volume was measured for 50 days. Tumorgrowth was observed every three days.

As shown in FIG. 8, tumor growth was significantly inhibited by MJ-66after 40 days (tumor volume, Control (N=7)=1007.3 mm³, DMSO(N=7)=922.5mm³, MJ-66 (N=8)=435.1 mm³, **p<0.01).

Even though numerous characteristics and advantages of the presentinvention have been set forth in the foregoing description, togetherwith details of the features of the invention, the disclosure isillustrative only. Changes may be made in the details within theprinciples of the invention to the full extent indicated by the broadgeneral meaning of the terms in which the appended claims are expressed.

What is claimed is:
 1. A compound of the formula I or a pharmaceuticallyacceptable salt, solvate or stereoisomer thereof:

wherein R₅, R₆, R₇ and R₈ independently represent H, OH, F, Cl, Br, C₁to C₆ alkyl group, C₁ to C₆ alkoxy group, C₂ to C₆ alkenyl group, C₂ toC₆ alkenoxy group, C₂ to C₆ alkynyl group, C₂ to C₆ alkynoxy group,amine group, mono- or di-substituted amino group, cyclic C₁ to C₅alkylamino group, imidazolyl group, morpholino group, piperazinyl group,optionally substituted with one or more hydroxy or halo; Ar represents

R₁′, R₂′, R₃′, R₄′, R₅′, R₆′, R₇′, R₈′ independently represent H, OH, F,Cl, Br, C₁ to C₆ alkyl group, C₁ to C₆ alkoxy group, C₂ to C₆ alkenylgroup, C₂ to C₆ alkenoxy group, C₂ to C₆ alkynyl group, C₂ to C₆alkynoxy group, amine group, mono- or di-substituted amino group, cyclicC₁ to C₅ alkylamino group, imidazolyl group, morpholino group,piperazinyl group, optionally substituted with one or more hydroxy orhalo; X represents NH, O or S.
 2. The compound as claimed in claim 1 ora pharmaceutically acceptable salt, solvate or stereoisomer thereof,wherein Ar is selected from the group consisting of naphthalenyl group,indolyl group and benzothiophenyl group.
 3. The compound as claimed inclaim 1 or a pharmaceutically acceptable salt, solvate or stereoisomerthereof, wherein R₆ is selected from the group consisting of: methoxylgroup, dimethylamino group, pyridinyl group, pyrrolidinyl group,piperidinyl group and morpholino group.
 4. The compound as claimed inclaim 1 or a pharmaceutically acceptable salt, solvate or stereoisomerthereof, which is:


5. A composition, which comprises a compound as claimed in claim 1 or apharmaceutically acceptable salt, solvate or stereoisomer thereof; and acarrier.
 6. The composition of claim 5, wherein Ar is selected from thegroup consisting of naphthalenyl group, indolyl group andbenzothiophenyl group.
 7. The composition of claim 5, wherein R₆ isselected from the group consisting of: methoxyl group, dimethylaminogroup, pyridinyl group, pyrrolidinyl group, piperidinyl group andmorpholino group.
 8. The composition of claim 5, wherein the compound is


9. A method for treating a disease or disorder comprising: administeringthe compound as claimed in claim 1 or a pharmaceutically acceptablesalt, solvate or stereoisomer thereof to a subject in need thereof. 10.The method of claim 9, wherein Ar is selected from the group consistingof naphthalenyl group, indolyl group and benzothiophenyl group.
 11. Themethod of claim 9, wherein R₆ is selected from the group consisting of:methoxyl group, dimethylamino group, pyridinyl group, pyrrolidinylgroup, piperidinyl group and morpholino group.
 12. The method of claim9, wherein the compound is:


13. The method as claimed in claim 9, wherein the disease or disorder isselected from the group consisting of: skin, lung, lymph node, breast,cervix, uterus, gastrointestinal tract, ovary, prostate, colorectal,mouth, brain, head and neck, throat, testes, kidney, pancreas, bone,spleen, liver, bladder, larynx and nasal passage cancers or tumors. 14.The method as claimed in claim 9, wherein the diseases or disorders aremalignant glioma, sarcoma, carcinoma or adenoma.
 15. The method asclaimed in claim 12, wherein the disease or disorder is selected fromthe group consisting of: skin, lung, lymph node, breast, cervix, uterus,gastrointestinal tract, ovary, prostate, colorectal, mouth, brain, headand neck, throat, testes, kidney, pancreas, bone, spleen, liver,bladder, larynx and nasal passage cancers or tumors.
 16. The method asclaimed in claim 12, wherein the diseases or disorders are malignantglioma, sarcoma, carcinoma and adenoma.